Antimicrobial Effect of Juice Extract From Fermented Cabbage Against Select Food- Borne Bacterial Pathogens

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1 Journal of Applied Sciences Research, 6(11): , , INSInet Publication Antimicrobial Effect of Juice Extract From Fermented Cabbage Against Select Food- Borne Bacterial Pathogens 1 Gogo L.A., 1 Shitandi A.A, 1 Lokuruka M.N.I., 2 Sang W 1 Department of Dairy and Food Science and Technology, Egerton University, P.O. Box , Egerton, Kenya 2 Centre for Microbiology Research, Kenya Medical Research Institute, P. O. Box Nairobi, Kenya Abstract: As part of ongoing efforts to use natural preservatives in meat preservation, this study investigated the antimicrobial effect of fermented cabbage juice against select food-borne bacterial pathogens. Unfiltered and filtered fermented cabbage (Brassica oleceracea L. var. capitata) juice were used for susceptibility testing using the dilution and agar well diffusion techniques. The screening was done against four standard bacterial strains (Salmonella enteritidis 13076, Escherichia Coli 25922, Staphylococcus aureus and Staphylococcus aureus 29213) and seven clinical isolates (Escherichia coli, Vibrio cholerae, Salmonella typhi, Salmonella typhimurium, Escherichia coli 0157; H7, Staphylococcus aureus and Salmonella enteritidis) of food borne bacterial pathogens. Pathogens grew well in juices from cabbage that was fermented for 28 days but were inhibited to varying extents in unfermented and fermented juices for up to 21 day cabbage fermentation. Activity was more pronounced against gram negative than gram positive bacteria. Inhibition was reduced, when the juice was filtered with 0.2 µm filter following centrifugation. The findings suggest that filtration and fermentation time could be factors responsible for activating a precursor into an inhibitory compound. It is concluded that fermented juice might be more effective against food-borne pathogens and spoilage bacteria when applied unfiltered and at day-21 ferments in meat. Key words: Brassica oleracea, Fermentation, Antimicrobial activity, Bacterial pathogens. INTRODUCTION The antibacterial activity of cabbage juice has been of interest to researchers with the activity been reported to be due to the glucosinolates degradation by-products found in the juice [1,2]. Cabbage juice has been shown to have antibiotic and antifungal activity against a wide range of bacteria, and has been used traditionally in the treatment of lung diseases [3,4]. Such activity has recently caused the need to revisit the potential of plant products as raw ingredients in the processing of other foods [5,6]. Cultivated plants with antibacterial activity would in general have more economic potential due to their grater adaptability to commercial propagation. Fermentation of the cabbage juice could enhance the bacteriostatic effect of the cabbage juice as acidified sodium chloride may be formed from the ferment [7]. Such juice may have potential for extended use in the biopreservation of other foods. There is however limited information on the antimicrobial activity of fermented cabbage juice extracts to further gauge its possibility as a bio-preservative. Therefore, in this study, the antimicrobial effect of fermented cabbage juice was determined against selected microbial pathogens. This was to gain a better understanding of the bio-preservative possibilities of the extracts is to be considered for harness in food systems. MATERIALS AND METHODS Fermentation of Cabbage and Juice Extraction: Fresh cabbage heads were randomly sourced from three sites markets in Kenya. The cabbages were shredded using a Black and Decker (Model FX750, Shelton, CT) Food Processing unit. Five hundred grams were weighed and common salt (Nacl) added at 2.25 %. The cabbage shreds were stacked in fermentation bottles in triplicate and left to ferment spontaneously at room temperature (approximately 20 o C). Juice samples were taken at day 0, 7, 14, 21 and 28 of fermentation for the inhibition experiments. For trials with filtered juices a 0.2 µm filter was used. Bacterial Strains and Culture Conditions: Eleven microbial strains were selected on the basis of their importance as food pathogens. Gram positive and gram Corresponding Author: Anakalo Shitandi, Department of Dairy & Food Science & Technology, Egerton University, Njoro, Box 536 Egerton 20115, Kenya. Phone ; Fax anakalos@gmail.com 1807

2 negative bacterial pathogens were obtained from culture collection maintained by the Kenya Medical Research Institute (KEMRI), Nairobi Kenya. Overnight cultures were prepared by transferring a loop full of stock cultures to tube having nutrient agar and incubating at 35 0 C for 24 h. Isolated colonies were used to prepare a bacterial suspension of 0.5 McFarland Standard 1 containing approximately 10 8 cfu/ ml. These cultures were then used as inoculums for culturing pathogens on petri dishes for the antimicrobial test. Water was used as negative control and ampicilin was used as positive control. Each sample was done in triplicate. Antibacterial activity was evaluated by quantifying zones of inhibition of bacterial growth after 24 hrs. Screening for Antibacterial Activity / Susceptibility Testing: The screening for antibacterial activity and susceptibility testing was done as described by Saeed & Tariq [1]. In brief, nutrient agar (NA) (Oxoid) was used as base medium for screening of antibacterial activity and tryptic soy broth (TSB) (Oxoid) for the preparation of inoculum. McFarland tube number 0.5 was prepared by mixing 9.95 ml of 1% (vol/vol) sulphuric acid and 0.05 ml of 1.175% barium chloride in distilled water in order to estimate bacterial density. This McFarland preparation gave a bacterial density of 10 8 cfu/ ml. The tube was sealed and used for comparison of bacterial suspension with the standard whenever required. Quality Control of Media and Reagents: Growth medium was prepared according to the manufacturer s instructions. After autoclaving, the medium was cooled to 50 C. About 25ml of medium per plate was measured into 15 x 100-mm plastic petri dishes on a level pouring surface to a uniform depth of 4 mm. Freshly prepared plates were used the same day or stored in a refrigerator (2 to 8 C) for up to 2 weeks. If plates were not used within 7 days of preparation, they were wrapped in plastic to minimize evaporation. Just before use, any excess moisture on the surface was removed by placing plates in an incubator (35 to 37 C) until the moisture evaporated (about 10 to 30 min). Each new lot was quality controlled before use by testing the E. Coli ATCC or Staphylococcus aureus ATCC standard strains, used with every test run for Enterobacteriaceae and gram-positive aerobes, respectively. Zone diameters obtained for ATCC were compared with the National Committee for Clinical Laboratory Standards (NCCLS) published limits [8]. Screening of antibacterial activity was performed by the well diffusion technique [1]. The nutrient agar plates were seeded with 0.1 ml of the standardized inoculum of each test organism. The inoculum was spread evenly over the plates with a sterile glass spreader. A standard cork borer of 8 mm diameter was used to cut uniform wells on the surface of the nutrient agar. Turbidity of the inoculum suspension was adjusted by holding the test tubes against a white background with contrasting line. A sterile cotton swab was dipped into the suspension used to pick colonies. The swab was streaked over the entire surface of the medium three times, rotating the plate approximately 60 degrees after each application to ensure an even distribution of the inoculum. About 200 μl of each juice of fermented cabbage was introduced in the well and allowed to stand for 15 minutes before incubation. The inoculated plates were incubated at 37 o C for 24 hours. After incubation, the diameter of the zones of complete inhibition were measured (including the diameter of the disk) and recorded in millimetres (mm). The scale of measurement was chosen as follows: $ 26 mm. Zone of inhibition - strongly inhibitory; 1-26 mm- moderately inhibitory; mm mildly inhibitory; < 11mm inhibitory to none inhibitory. The working supply of antimicrobial disks stored in the refrigerator (4 C) was used as positive control to evaluate cultures for possible antibiotic resistance patterns that would have affected the sensitivity of assay. Microbial sensitivity disks (Oxoid, UK) for the drug resistance patterns used were gentamicin (G) (10 μg/disk), ciprofloxacin (Cp) (5 μg/disk) and nalidixic acid (Na) (30 μg/disk). Upon removal of the disks from the refrigerator, the package containing the cartridges was left unopened at room temperature for approximately 1 hour to allow the temperature to equilibrate to room temperature before using. This reduced the amount of condensation on the disks. The disks were placed individually with sterile forceps, and then gently pressed down onto the inoculated agar. After the disks were placed on the plate, the plates were inverted and incubate at 37 C for hours. The distance from the colonies closest to the disk centre was measured and then doubled to obtain a diameter. Minimum Inhibitory Concentrations of The Juices: The serial dilution technique was used to test levels of diluted juice that could cause inhibition to selected pathogens. Juices were serially diluted using sterile distilled water to levels of up to 1: The wells so formed on pathogen inoculated plates were filled with diluted juices on plates at portions of 0.2 ml then incubated at 37 0 C for 24 hrs. Plates were then observed for inhibition zones up to the lowest dilution where inhibition occurred. This dilution was recorded as the minimum inhibitory concentration. 1808

3 The Growth Experiment: Juices were added at replacement values of 0%, 25%, 50%, 75%, and 100% respectively to sterile tryptic soy broth, inoculated with 100 µl of 0.5 McFarland standard culture containing 10 8 cfu/ ml of organisms approximating to a culture of 10 7 cfu/ ml. This was incubated for 24 hrs at 37 0 C. After incubation sample turbidity was measured using a spectrophotometer and absorbance readings recorded for each sample. A sample of 100% represented microbial growth on juice alone and 0% represented growth on media broth without added juice. Growth Inhibition Experiments: Response surfaces methodology for the combined effect on growth of temperature, day of fermentation and sterile media dilutions with fermented juice were studied at four levels each. The double dilution method was used. About 10 ml of juices from Kinale that were fermented for 0, 7, 14 and 21 days were added to 4 prepared tubes of 10 ml sterile tryptic soy broth to give 0.5, 0.25, and The dilutions were inoculated with 0.1 ml of 0.5 McFarland standard culture containing 10 7 cfu/ ml of three standard ATCC bacterial strains. The culture was the incubated at 4 0 C, 22 0 C, 37 0 C and 42 0 C respectively. After 24 hr incubation the optical density was recorded to obtain the growth responses. Trials with Pathogen Contaminated Meat Samples: Lean upper cut meat weighing 2 kg was purchased from a local supermarket. Blocks of meat measuring 2.5 x 2.5 by 1.25 cm were incised from the meat using a knife. Then, 0.1 ml of inoculum was added drop wise to the beef cube and distributed by overturning using a sterile glass spreader. After a 30-min holding period at room temperature to allow cell attachment, the cubes were dipped in 150 ml fermented cabbage juices of day 14 and 28. The dipped cubes were placed under refrigeration at 4 0 C for 4 days. After 4 days the cubes were removed from the juice and washings made using 250 ml of physiological saline. The saline with beef was shaken for 30 s then, 1 ml aliquots of the wash was inoculated on spread plates of selective agar, incubated at 37 0 C and growth observed after 24 hr. Statistical Analysis: Experiments were performed in triplicate and diameters of zones of inhibition were expressed as mean ± standard deviation. Where the main effect was significant (P < 0.05) means were separated using the least significant difference (LSD) test. Response surface methodology was used for experimental design, data analysis and model building using SAS (Version 8.2, SAS Inst. Inc., Cary, N.C., USA) for the combined effect of three hurdles on growth of three microbial strains. Three independent variables vis a vis temperature, day of juice fermentation and level of media dilution with the juice, with four levels for each variable were used, while the independent variable was growth which was taken from absorbance readings. Responses were considered significant at p < RESULTS AND DISCUSSION The results of susceptibility testing with unfiltered and filtered juices from fermented cabbage are presented in table 1 and fig 4. Both unfiltered and filtered juices showed antimicrobial activity against the tested food-borne pathogens to varying extents with the unfiltered juices having a greater effect. Sulphur compounds from cabbage have been found to show strong antimicrobial activity [9]. Fermentation does not seem to impair the antimicrobial activity suggesting that the activated precursors into inhibitory compounds persist during the process. The highest activity observed in juices from cabbage fermented for 21 days for all the three locations was observed in the clinical strains of V. cholerae, S. enteritidis, and S. typhi while the lowest activity was observed in tests with E. coli ATCC This seems to coincide with the known quality values for sauerkraut which has been observed after day 21 fermentation periods [9]. The amounts of fermentable sugars and ph are lowest at this point with highest titratable acidity as percent lactic acid [9]. Moderate to slight inhibition of the pathogens was observed with juices from cabbages fermented for 7 and 14 days respectively indicating that day 7 day ferments showed better effect than day 14 ferments. The susceptibility of individual bacterial strains to unfiltered juice from fermented cabbage was varied. The descending order of the juice antimicrobial activity was S. Enteritidis 13076, S. aureus 43300, S. aureus and E. coli for the ATCC strains. A stronger antimicrobial activity of the juice was observed on the clinically isolated strains compared to the laboratory strains. The pathogen most resistant to antimicrobial activity of the studied juices was E. coli This observation was similar to that made with the filtered juices on the same organism. Escherichia coli has been shown to offer a higher antimicrobial resistance in other studies compared to other strains [10]. There was a significant difference in the activity of the juice on the individual standard strains studied (table 1). A mild inhibition with mean zones of 13.1 mm, 11.7 mm, 13.9 mm and 12.9 mm for S. Enteritidis 13076, E. coli 25922, S. aureus and S. aureus 29213, respectively were recorded. Inhibitory activity was the same for S. Enteritidis and S. aureus on separation of treatment means. The highest growth inhibition was observed on S. aureus and 1809

4 lowest on E. coli ATCC strains. Staphylococcus aureus has previously been found to be more susceptible than E. coli strains in studies with spices used in meat processing [11]. Inhibition increased with increasing days of fermentation. Juice from cabbage fermented for 21 days was observed to have the highest mean inhibition for all the four ATCC strains irrespective of geographic location, except for day-21 ferments from Dundori which exhibited strongest inhibition on all strains studied. In a different study where several spices were studied only one spice showed highest antimicrobial activity on all strains studied [11]. This inhibition as shown in table 2 was however eliminated in the juice from unfermented cabbage and 7 days fermentation when the juice was filtered for all the organisms. The results of tests with meat samples are presented in table 3. No growth was observed in the selective media used for growth of the tested strains after hrs. This growth inhibition could have been due to a number of antimicrobials in the juice. Lactic acid bacteria in ground meat has been shown in a precious study to inhibit the growth of E. coli 0157:H7 in ground beef [13]. The reduction of ph during fermentation has further been shown to cause the protection of ground pork against pathogens [13,14, 15]. In this study while some bacterial strains were inhibited with undiluted juices inhibition was generally observed after dilution of 1:10 3 on both ATCC and clinical isolates of S. aureus strains (Table 3). The results of this study are different from what was observed with cabbage juices in a similar study [16]. However, fermented juices in dilutions of 1:1 and 1:5 showed dose related effects. Allyl isothiocyanates in sauerkraut have been shown to inhibit Salmonella typhimurium and E. coli [9,17] although this has to be treated cautiously as many factors in food systems including buffering of proteins, emulsifier effects, phospholipids and fatty acids cause masking in the food matrix [18]. The linear dependence of absorbance on the number of microorganisms in CFU/ml was observed in figures 1-3. Day 7 and 21 showed the highest inhibition to growth. Inhibition to growth was mainly due to the effect of temperature and day of fermentation minimum inhibition being exerted at 37 0 C for S. enteritidis at the seventh day of fermentation (table 1). The critical temperature found in this work was being 42 0 C on the 7 th day. The findings indicate that for effective antibiotic effect, juice fermentation must proceed beyond 7 days. The level of juice dilution studied were found to cause an insignificant (p > ) effect on growth for this organism. Table 1: Susceptibility of standard American Type Culture Collection (ATCC) pathogenic bacterial strains to juice from fermented cabbage Inhibition zone diameter in mm (Mean* ± SD) Juice type UNFILTERED JUICES FILTERED Site / Day D 0 17 ± 4.36 e ± 1.53 bcd 16 ± 3.46 efg 13 ± 1.00 b R R R R M0 16 ± 6.08 e ± 0.58 bcd ± 0.58 h 13 ± 1.00 b R R R R K0 16 ± 5.29 e 11 ± 0.00 cde ± 0.58 def 12 ± 1.73 b R R R R D7 17 ± 2.64 e 9.33 ± ef ± 1.53 fgh 12 ± 2 00 b R R R R M7 22 ± 5.29 bc 14 ± 1.00 abc 25 ± 4.36 b ± 1.15 a 13.5 ±2.12 d R R 12.5 ± 0.71 c K ± 5.85 e ± 1.53 def ± 1.53 de ± 0.58 b R R R R D14 16 ± 3.61 e R ± 0.58 gh ± 0.58 b 13 ± 2.83d 14 ±1.41 b 15.5 ± 0.71 d 13.5 ± 0.71 c M ± 3.06 cd 17 ± 1.00 a ± 1.53 cd 17 ± 1.00 a 15.5 ± 0.71c 14 ± 0.00 b 20.5 ± 0.71 bc 17.5 ± 0.71 b K ± 0.58 de 12 ± 0.00 cde 17 ± 1.00 ef 15 ± 1.00 ab 15.5 ± 0.71c 11.5 ± 0.71 cd 19.5 ± 0.71 c 13.5 ± 0.71 c D21 24 ± 4.58 ab ± 0.58 ab 21 ± 1.73 c ± 0.58 a 17.5 ± 0.71 b 19 ± 0.00 a 21.5 ± 0.71 b 20.5 ± 0.71 a K21 23 ± 2.65 bc 17 ± 1.00 a 23 ± 1.00 bc 18 ± 1.00 a 18 ± 0.00 b 15 ± 0.00 b 20.5 ± 0.71 bc 17.5 ± 0.71 b M ± 1.52 a R ± 1.15 a ± 0.58 a 17.5 ± 0.71 b 12 ± 0.00 c 16.5 ± 0.71 d 18.5 ± 0.71 b D ± 2.31 f R ± 1.53 efg ± 0.58 b 17.5 ± 0.71 b 14 ±1.41 b 15.5 ± 0.71 d 12.5 ± 0.71 c M28 R R 16 ± 4.58 efg R 21.5 ±2.12 a 18 ±1.41 a 23.5 ± 0.71 a 20.5 ± 0.71 a K28 R R ± 0.58 h R R R R R * Means of 3 determinations R- Zone diameter 8 ± 0.00 indicating resistance abc - means followed by the same letter along the same column are not significantly different 1- S. Enteritidis E. coli S. aureas S. aureas D- Dundori M- Molo K - Kinale 0, 7, 14, 21, 28 - Days of fermentation 1810

5 Table 2: Susceptibility of clinically isolated pathogenic bacterial strains to fermented cabbage juice Inhibition zone diameter in mm (Mean* ± SD) Site / Day Strain D 0 11 ± 1.41 cd 14.4 ± 0.71 fg 12.5 ± 0.71 cd R 15 ± 1.41 abc 12.5 ± 0.71 e 17 ± 0.00 dcd M ± 0.71 ab 16.5 ± 0.71 ef 12 ± 1.41 gh 12.5 ± 0.71 bc 14.5 ±3.54 abc 13.5 ± 2.12 de 18 ± 1.41 bcd K0 11 ± 1.41 cd 14.5 ± 0.71 fg 13.5 ± 0.71 efg 10.5 ± 0.71 cd 13 ± 1.41 c 14 ± 1.41 cde 14 ± 1.41 de D7 19 ± 1.41 a 18.5 ± 0.71 cdef 14 ± 1.41 defg 10.5 ± 0.71 cd 14 ± 1.41 bc 12.5 ± 0.71 e 16.5 ± 0.71 a M7 12 ± 0.00 cd 18 ± 1.41 def 17 ± 0.00 cde 10.5 ± 0.71 cd 12.5 ± 0.71 c 13 ± 1.41 e 17.5 ± 0.71 bcd K7 18 ± 1.41 a 23.5 ± 0.71 ab 18 ± 1.41 cd 12.5 ± 0.71 bc 18.5 ± 0.71 a 17.5 ± 0.71 dcd 20 ± 2.83 b D ± 0.71 a 22 ± 1.41 abcd 21 ± 1.41 bc 13 ± 1.41 bc 13 ± 1.41 c 12.5 ± 0.71 e 21 ± 2.83 b M ± 0.71 ab 22.5 ± 4.95 abc 25 ±1.41b 12.5 ± 0.71 bc 13 ± 1.41 c R 19.5 ± 0.71 b K ± 0.71 bc 17.5 ± 0.71 ef 23.5 ± 0.71 b 13.5 ± 2.12 abc 14.5 ± 2.12 abc 13.5 ± 0.71 de 15 ± 1.41 cd D ± 2.12 a 20 ± 2.83 bcde 24.5 ± 0.71 b 13.5 ± 2.12 abc 16.5 ± 0.71 abc 16.5 ± 0.71 bcde 18.5 ± 0.71 bc K21 21 ± 2.83 a 25 ± 1.41 a 23 ± 5.66 b 16.5 ± 0.71 ab 18.5 ± 0.71 a 18 ± 1.41 bc 18.5 ± 0.71 bc M21 18 ± 7.07 a 22.5 ± 4.94 abc 29.5 ± 0.71 a 15.5 ± 0.71 ab 14 ± 1.41 bc 19.5 ± 0.71 b 15.5 ± 0.71 a D28 R 14.5 ± 0.71 fg 17 ± 2.83 cde 13.5 ± 0.71 abc 17.5 ± 1.53 ab 16.5 ± 0.71 bcde 18 ± 1.41 bcd M28 R 18.5 ± 4.91 cdef 16.5 ±2.12 def 17.5 ± 0.71 a 17.5 ±2.12 ab 25 ± 1.41 a 19 ± 1.41 bc K28 R 11.5 ± 0.71 g R 10.5 ± 0.71 cd R R 10 ± 0.00 e * Means of 3 determinations R- Zone diameter 8 ± 0.00 indicating resistance abc - means followed by the same letter along the same column are not significantly different 5- E. coli 6- V. cholerae 7- S. typhi 8- S. typhimurium 9- E coli 0157:H7 10- S aureas 11 -S. enteritidis Table 3: The canonical analysis for the estimated stationary points and overall curve shapes of the growth responses of the bacterial strains. Eigen vectors critical values Bacterial Strain Eigen values Day Dilution Temperature Stationary point Uncoded Coded S. enteritidis Day Saddle point Dilution Temperature E. coli Day Saddle point Dilution Temperature S. aureas Day maximum point Dilution Temperature

6 Fig. 1: Scatter diagram for effect of fermented cabbage juice from D (Dundori) on the growth of the three ATCC strains. Q1- S. Enteritidis 13076, Q2- E coli 25922, Q4- S aureas D7- Day 7 fermentation, D21- Day 21 fermentation. Fig. 2: Scatter diagram for effect of fermented cabbage juice from M (Molo) on the growth of the three ATCC strains. Q1- S. Enteritidis 13076, Q2- E coli 25922, Q4- S aureas D7- Day 7 fermentation, D21- Day 21 fermentation. Fig. 3: Scatter diagram for effect of fermented cabbage juice from K (Kinale) on the growth of the three ATCC strains. Q1- S. Enteritidis 13076, Q2- E coli 25922, Q4- S aureas D7- Day 7 fermentation, D21- Day 21 fermentation. 1812

7 The day of juice fermentation and incubation temperature from table 1, was observed to significantly (p< ) affect the growth response of S. enteritidis. Overall in this study the activity of the juice was found to be dependent on the bacterial strain and temperature of incubation. The organosulfur components methyl methanethiosulfinate and s-methyl -L- cysteine sulfoxide and sulphides have been found in unfermented cabbage to have antimicrobial effects on some strains [9]. These compounds were however not determined in this study but they might be able to persist during the process of fermentation. It has been shown that organic acids found in fermented foods can inhibit a wide range of microorganisms [19,20]. Conclusion: The results of this study indicate that the strongest inhibitory activity was found in fermented cabbage juices after 21-day fermentation. The unfiltered juice extract further had the higher level of inhibition. In optimising the use of the fermented vegetable the juice could thus be best after a 21 day fermentation period and unfiltered for it to be used as a preserving ingredient in meat curing for the studied microbes. ACKNOWLDGMENT We are grateful to the German development agency (DAAD) who provided financial support for this research. We also thank and the Kenya Medical Research Institute (KEMRI) for kindly availing the research facilities which enabled this work to be done. REFERENCES 1. Saeed, S. and P. Tariq, Effects of some seasonal vegetables and fruits on the growth of bacteria. Pakistan Journal of Biological Sciences, 9(8): Stoewsand, G.S, Bioactive organosulfur phytochemicals in Brassica oleracea vegetables. A review. Food Chemistry and Toxicology, (33): Chopra, D. and D. Simon, D, The Chopra Centre Herbal Handbook. Rider, London. 4. Dutta, K., I. Rahman and K. Das, Antifungal activity of Indian plant extracts. Mycoses, (41): Sebranek, J.G. and J. Bacus, Cured meat products without direct addition of nitrate or nitrite: what are the issues? Meat Science, (77): Honikel, K.-O, The use and control of nitrate and nitrite for the processing of meat products. Meat Science, (78): Intsu, Y, Efficacy of Acidified Sodium Chlorite treatment in reducing E. Coli 0157:H7 on Chinese Cabbage. J. Food. Prot., (68): National Committee for Clinical Laboratory Standards (NCCLS), Performance Standards for Antimicrobial Disk Susceptibility Tests. Approved Standard., 6th ed. Wayne, Pa, National Committee for Clinical Laboratory standards, M2- A6. 9. Kyung, K.H. and H. Fleming, Antimicrobial activity of sulphur compounds derived from cabbage. Journal of Food Protection, 60(1): Oliver, M. Nunez and S. Gonzalez, Fermented foods: Fermented vegetable products. In : Encyclopedia of food microbiology. (Eds.) Robinson R. K., Ball C. A., & Palel D. D Press, Sema, A., D. Nursel and A. Suleyman, Antimicrobial activity of some spices used in the meat industry. Bulletin of Veterinary Institute, Pulaway, (51): Agaoglu, S., N. Dostbil and S. Alemdar, Antimicrobial activity of some spices used in the meat industry. Bulletin. Veterinary Institute Pulawy., (51): Vold, L., Y. Wasteson and V. Nissen, High levels of background flora inhibits growth of Escherichia coli O157:H7 in ground beef. International Journal of Food Microbiology., (56): Sakhare, P., Z. Narasimha and D. Rao, Microbial profiles during lactic fermentation of meat by combined starter cultures at high temperatures. Food Control, (14): Minor-Pérez, H., E. Ponce-Alquicira, S. Macías- Bravo and I. Guerrero-Legarreta, Changes in fatty acids and microbial populations of pork inoculated with two biopreservative strains. Meat Science, (66): Tolonen, M., The formation and antibacterial activity of nisin and plant derived bioactive compounds in lactic acid bacteria fermentations. Doctoral dissertation. Agrifood research reports Finland, (43): Shofran, G., T. Purrington, F. Breidt and P. Fleming Antimicrobial activity of sinigrin and its hydrolysis products. Journal of Food Science, (63): Mazzotta, S. and J. Montville Nisin induces changes in membrane fatty acid composition of Lysteria monocytogenes nisin-resistant strains at 10 0 C and 30 0 C. Journal of applied Microbiology, (82): Nout, R. and M. Rombouts, Fermentative preservation of plant foods. Journal of Applied Bacteriology. Symposium Suppliment, (73): Cardrici, B. and S. Citak S A comparison of two methods used for measuring antagonistic activity of lactic acid bacteria. Journal of Nutrition, 4(4):